The present invention relates to monovalent and multivalent RNA aptamers, constructed DNA molecules and engineered genes which encode the RNA aptamers of the present invention, as well as expression systems, host cells, and transgenic organisms which express the RNA aptamers of the present invention.
Cells and organisms are complex adaptive systems in which numerous biological processes are driven by sophisticated macromolecular machinery and regulated by elaborate signal transduction networks, both usually composed of multiple proteins. To better understand and control such processes, new technologies are needed to intervene in protein functions in the real time and space of the living cells. In many cases, such in vivo destructive approaches are needed to expand and extend results obtained from in vitro reconstruction studies. On the other hand, many diseases are known to be caused by either overexpression of certain endogenous genes (such as oncogenes in cancer) or expression of exogenous genes (as in the case of a virus infection), and xe2x80x9canti-genexe2x80x9d therapies are called for to avert or ameliorate the morbidity and mortality caused by these gene products. To inactivate a specific gene or gene product, different techniques are directed at three distinct types of targets: DNA, RNA, and protein. For example, a gene can be altered by homologous recombination, the expression of the genetic code can be blocked at the RNA level by antisense oligonucleotides or ribozymes, and the protein function can be altered or inhibited by antibodies or drugs.
A particularly useful tool resulting from the change of the protein coding function of genes is a conditional allele which displays its mutant phenotype only under certain non-permissive conditions, making it possible to obtain viable cells or organisms when a critical protein is under investigation. More importantly, with a conditional allele it is also possible to target and change specific genes in specific stages of development so that the details of a wrongly assembled protein machine can be identified. Recently there have been many new refinements of this technique. Notably, Struhl and colleagues developed a two-pronged approach to create yeast strains with conditional alleles in which the addition of copper ion leads to the simultaneous cessation of MRNA synthesis and destruction of the target protein in the cell (Moqtaderi et al., xe2x80x9cTBP-Associated Factors Are Not Generally Required for Transcriptional Activation in Yeast,xe2x80x9d Nature 383:188-191 (1996)). However, the generation of conditional mutants in higher (i.e., multicellular) eukaryotes is quite difficult. In addition, it is often impossible to assay individual domains or discrete functional surfaces of a protein, since the function of the whole protein is abolished.
Small molecular mass drugs and drug derivatives that directly target proteins have been used not only clinically to rectify disease phenotype, but also in basic research that yielded ample information in mechanistic studies both in vitro and in vivo. These are usually cell-permeable, low molecular weight organic molecules identified from natural sources or designed and synthesized in the laboratory. Usually they are specific ligands of proteins, affecting protein functions upon binding. In many cases they are mimetics of the natural ligands of their targets (or receptors, as they are called in pharmacodynamics). In vivo experiments can be conducted easily with drugs at the cellular level since the administration may be simple diffusion governed by Fick""s law. But systemic drug delivery to the organism is usually complicated by many pharmacokinetic factors, making it difficult to institute dosage regimens and assess drug effects at high temporal-resolution. The biggest limitation of using small molecular protein ligands is their availability. It is usually not easy to find such a ligand for a predetermined protein target, either from natural sources or by design. Recently, a general procedure for manipulating protein in vivo at the cellular level was developed, in which a gain of function results from the use of synthetic xe2x80x9cdimerizersxe2x80x9d derived from an immunosuppressive drug (Ho et al., xe2x80x9cDimeric Ligands Define a Role for Transcriptional Activation Domains in Reinitiation,xe2x80x9d Nature 382:822-826 (1996)). Although this xe2x80x9cthree-part inventionxe2x80x9d (Crabtree and Schreiber, xe2x80x9cThree-Part Inventions: Intracellular Signaling and Induced Proximity,xe2x80x9d TIBS 21:418-422 1996)) may overcome the difficulty to a certain extent, a ligand-binding domain has to be appended to the target proteins.
As specific protein binding ligands, antibodies can be custom-made for virtually any given protein, due to the clonal selection and maturation function of the immune system. Antibodies raised against specific proteins have made possible many technological advances in the field of molecular biology, including modern immunochemistry (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)). But the in vivo utility of protein reagents like antibodies is severely limited by difficulties in their delivery and their own immunogenicity.
RNA has distinct advantages over proteins and small organic molecules when considering its use to inactivate protein function in vivo. An RNAencoding sequence can be linked to a promoter and this artificial gene introduced into cells or organisms. Depending on the regulatory sequence included, this provides a unique way of constructing a time and/or tissue specific suppresser gene. Such RINA expressing genes are usually smaller than protein-coding genes and can be inserted easily into gene therapy vectors. Unlike a foreign or altered protein, RNA is less likely to evoke an immune response. Antisense molecules and ribozymes have been developed as xe2x80x9ccode blockersxe2x80x9d to inactivate gene function, with their promise of rational drug design and exquisite specificity (Altman, xe2x80x9cRNase P in Research and Therapy,xe2x80x9d Bio/Technology 13:327-329 (1995); Matteucci and Wagner, xe2x80x9cIn Pursuit of Antisense,xe2x80x9d Nature 384 Suppl. (6604):20-22 (1996)). Mechanistically, both antisense oligodeoxynucleotides (xe2x80x9cODNsxe2x80x9d) and bioengineered ribozymes are expected to achieve specific binding in the first step of their action by forming a stable duplex (or triplex in some cases of the ODNs) with a target nucleotide sequence based on Watson-Crick or Hoogsteen base pairing. However, this mechanism and their ability to disrupt the function of a single gene has never been proven. Furthermore, a wide variety of unexpected non-antisense effects have come to light, especially with the chemically modified compounds. Although some of these side effects may have clinical value, the use of antisense compounds as research reagents is severely limited (Branch, xe2x80x9cA Good Antisense Molecule is Hard to Find,xe2x80x9d TIBS 23:45-50 (1998)).
Recently, RNA aptamers have also been explored as research and therapeutic reagents for their ability directly to disrupt protein function. Selection of aptamers in vitro allows rapid isolation of extremely rare RNAs that have high specificity and affinity for specific proteins. Exemplary RNA aptamers are described in U.S. Pat. No. 5,270,163 to Gold et al., Ellington and Szostak, xe2x80x9cIn vitro Selection of RNA Molecules That Bind Specific Ligands,xe2x80x9d Nature 346:818-822 (1990), and Tuerk and Gold, xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase,xe2x80x9d Science 249:505-510 (1990). Unlike antisense compounds, whose targets are one dimensional lattices, RNA aptamers can bind to the three dimensional surfaces of a protein. Moreover, RNA aptamers can frequently discriminate finely among discrete functional sites of a protein (Gold et al., xe2x80x9cDiversity of Oligonucleotide Functions,xe2x80x9d Annu. Rev. Biochem. 64:763-797 (1995)). As research and therapeutic reagents, aptamers not only have the combined advantages of antibodies and small molecular mass drugs, but in vivo production of RNA aptamers also can be controlled genetically. The controlled expression of high affinity RNA aptamers offers a means of rapidly inactivating specific domains of proteins and thereby assessing their function and mechanism of action.
Although gene therapy has the potential for treating many diseases with very low risk of adverse reactions, the efficiency of gene transfer and expression in vivo is still disappointingly low. Assuming that efficient gene transfer can be developed, the next issue would be long-term, stable, or even regulated gene expression at the appropriate level. This is perhaps the greatest shortcoming of present vectors for gene therapy (Anderson, xe2x80x9cHuman Gene Therapy,xe2x80x9d Nature 392 Suppl. (6679): 25-30 (1998)). Efficient and effective intracellular expression of functional RNA molecules such as aptamers depends on many factors, some of them giving rise to competing and conflicting design requirements. Ideally, the RNA should be productively transcribed, stabilized against rapid degradation, folded correctly, and directed to the subcellular region where its target resides. Genes expressing various inhibitor RNAs have been generated by modifying small RNA transcription units that normally produce tRNAs (Sullenger et al., xe2x80x9cOverexpression of TAR Sequences Renders Cells Resistant to Human Immunodeficiency Virus Replication,xe2x80x9d Cell 63:601-608 (1990)), small nuclear RNAs (Noonberg et al., xe2x80x9cIn vivo Generation of Highly Abundant Sequence-Specific Oligonucleotides for Antisense and Triplex Gene Regulation,xe2x80x9d Nucleic Acids Res. 22:2830-2836 (1994)), or small viral RNAs (Lieber and Strauss, xe2x80x9cSelection of Efficient Cleavage Sites in Target RNAs by Using a Ribozyme Expression Library,xe2x80x9d Mol. Cell. Biol. 15:540-551 (1995)). Although high level RNA accumulation has been achieved in some cases, a major disadvantage of such transcription units is the limited ability to regulate their expression. Also, tRNA promoters have intragenic promoter elements, resulting in RNA transcripts carrying additional tRNA sequence which may affect the folding of the adjoining functional RNA moiety.
The present invention is directed to overcoming these and other deficiencies in the art.
As used herein, the term xe2x80x9captamerxe2x80x9d refers to reagents generated in a selection from a combinatorial library (typically in vitro) wherein a target molecule, generally although not exclusively a protein or nucleic acid, is used to select from a combinatorial pool of molecules, generally although not exclusively oligonucleotides, those that are capable of binding to the target molecule. The selected reagents can be identified as primary aptamers. The term xe2x80x9captamerxe2x80x9d includes not only the primary aptamer in its original form, but also secondary aptamers derived from (i.e., created by minimizing and/or modifying) the primary aptamer. Aptamers, therefore, must behave as ligands, binding to their target molecule.
One aspect of the present invention relates to a monovalent RNA aptamer that binds to Drosophila splicing factor B52.
Another aspect of the present invention relates to a multivalent RNA aptamer that includes at least two RNA aptamer sequences linked together.
Yet another aspect of the present invention relates to an isolated or constructed DNA molecule encoding either a monovalent RNA aptamer or a multivalent RNA aptamer of the present invention.
Still another aspect of the present invention relates to an engineered gene encoding a multivalent RNA aptamer, where the engineered gene includes a DNA sequence encoding a multivalent RNA aptamer and a regulatory sequence which controls expression of the DNA sequence encoding a multivalent RNA aptamer.
Another aspect of the present invention relates to a method of expressing a multivalent RNA aptamer in a cell which includes introducing either a DNA molecule or an engineered gene of the present invention into a cell under conditions effective to express the multivalent RNA aptamer.
Yet another aspect of the present invention relates to a method of inhibiting activity of a target molecule in a cell which includes expressing a multivalent RNA aptamer in the cell, the multivalent RNA aptamer having an affinity for a target molecule sufficient to inhibit activity of the target molecule.
Another aspect of the present invention relates to a method of increasing activity of a splicing factor protein in a cell. This method includes inserting a multivalent RNA aptamer, which binds to a splicing factor protein, into an RNA transcript, which contains exons and introns, under conditions effective to enable splicing of the RNA transcript.
A further aspect of the present invention relates to a transgenic non-human organism whose somatic and germ cell lines contain an engineered gene encoding a multivalent RNA aptamer which inhibits activity of a target molecule to treat a condition associated with an expression level of the target molecule.
Additional aspects of the present invention include a constructed DNA molecule that contains a plurality of monomeric sequences each encoding a functional RNA molecule; an engineered gene that includes a DNA sequence containing a plurality of monomeric sequences each encoding a functional RNA molecule and a regulatory sequence which controls expression of the DNA sequence; and a transgenic non-human organism whose somatic and germ cell lines contain an engineered gene encoding a functional RNA molecule, where the functional RNA molecule inhibits the activity of a target molecule to treat a condition associated with an expression level of the target molecule.
Still further aspects of the invention relate to methods of expressing a functional RNA molecule in a cell by introducing either a constructed DNA molecule or an engineered gene, which encode the functional RNA molecule, into a cell under conditions effective to express the functional RNA molecule.
By coupling in vitro selection with in vivo transcriptional regulation, a multivalent RNA aptamer can be constructed that has a higher affinity for its target molecule (e.g., protein, nucleic acid, etc.) than its component RNA aptamers. When its in vivo transcription is regulated, the multivalent RNA aptamer of the present invention can be used according to a general methodology to inhibit in vivo functions of a specific target molecule. As shown herein using the Drosophila splicing factor protein B52 as a model system, a multivalent RNA aptamer of the present invention, when expressed in cells of cell culture or in somatic and germ cells of a transgenic organism, can act as a protein antagonist in vivo. When the multivalent RNA aptamer is expressed in the somatic and germ cells of a transgenic organism, activity of the target protein is inhibited to treat a condition associated with an expression level of the target protein. The multivalent RNA aptamers of the present invention have the combined advantages of prior art systems described above, but it eliminates their major shortcomings. Like antibodies, the multivalent RNA aptamers can be made to inhibit activity of specific target proteins. Like small organic molecules, multivalent RNA aptamers can directly target specific domains or discrete functional surfaces of the target protein within cells. Like conditional alleles, administration and expression of the multivalent RNA aptamers can be controlled genetically in whole organisms. In addition, expression of the multivalent RNA aptamers can be limited to specific tissues, cells, or stages of development.